Endophytic yeast strains, methods for ethanol and xylitol production, methods for biological nitrogen fixation, and a genetic source for improvement of industrial strains

ABSTRACT

The present invention provides novel endophytic yeast strains capable of metabolizing both pentose and hexose sugars. Methods of producing ethanol and xylitol using the novel endophytic yeast are provided herein. Also provided are methods of fixing nitrogen and fertilizing a crop using the novel endophytic yeast strains provided herein.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application No. 61/160,077 filed Mar. 13, 2009, expressly incorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK

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BACKGROUND OF THE INVENTION

Endophytic microorganisms occur within living plant tissues without causing apparent damage to the host (Petrini, 1991). To date, endophytic yeasts have been isolated from a variety of plants, including roots of Zea mays L. (maize) (Nassar et al., 2005) and roots of Musa acuminate L. (banana) (Cao et al., 2002), and leaves of Oryza sativa L. (rice) (Tian et al., 2004), Solanum lycopersicum L. (tomato) (Larran et al., 2001), and Triticum aestivum L. (wheat) (Larran et al., 2002). In a series of studies on endophytic microorganisms in wild and hybrid Populus species, (Doty et al., 2005; Doty et al., 2009) three yeast strains were isolated.

Identification of yeast species from morphological and physiological characteristics has been complemented with and improved by molecular methods in the last 20 years. Analyses of small subunit (18S) ribosomal RNA (rRNA) gene sequences, extremely important in phylogenetic analyses of species in bacteria, generally are not adequate to differentiate yeast species (James et al., 1996; Kurtzman and Robnett, 2003). Sequencing domains 1 and 2 (D1/D2) of large subunit (26S) rRNA gene have been used by many researchers to determine yeast species because this approach is rapid and effective, and a large number of sequences are available for comparison in online databases (Kurtzman and Robnett, 1998; Fell et al., 2000; Kurtzman, 2006). The internal transcribed spacer (ITS) regions ITS1 and ITS2, flanking the 5.8S gene of rRNA, are also highly substituted and are used for yeast identification. Scorzetti et al. (2002) found that analyzing ITS sequences allowed them to detect species among basidiomycetous species more effectively than using D1/D2. For example, Sporobolomyces holsaticus Windisch ex Yarrow & Fell and Sporidiobolus johnsonii Nyland are identical in D1/D2 sequences, register 93% DNA hybridization (Boekhout, 1991), and differ in five base positions in the ITS sequences. In contrast, Rhodotorula glutinis (Fresen.) F. C. Harrison and Rhodotorula graminis Di Menna are identical in the ITS region but differ in one base position in D1/D2; they are considered to be separate species based on 35-40% DNA hybridization (Gadanho and Sampaio, 2002). Consequently, it appears useful to sequence both D1/D2 and ITS regions when distinguishing closely related species, while defining species taxonomically requires classical phenotypic information (Scorzetti et al., 2002).

Recently, the role of endophytic microorganisms in the promotion of plant growth has received increased attention. Endophytes can promote plant growth through different mechanisms, including delivery of fixed nitrogen to host plants, production of plant growth regulators, and biological control of plant pathogens (Ryan et al., 2008). Endophytic yeast strains have been shown to be able to promote the growth of maize (Nassar et al., 2005) and Beta vulgaris L. (sugar beet) (El-Tarabily, 2004) by producing plant auxins, such as indole-3-acetic acid (IAA) and indole-3-pyruvic acid (IPYA) (Nassar et al., 2005).

Efficient industrial production of biofuels, such as bioethanol, holds promise for serving the growing energy needs of the world in the near future. Ethanolic fermentation of cellulosic and lignocellulosic biomass by microorganisms such as yeast is currently employed in the industrial production of bioethanol. However, the lack of non-pathogenic microorganisms that efficiently metabolize both five carbon (pentose) and six carbon (hexose) sugars in the presence of high levels of ethanol, limits the efficiency and therefore the economic feasibility of large-scale fermentations of certain lignocellulosic carbon sources that contain high levels of hemicellulosic biomass. As an example of this, in the absence of corn, the maize plant is comprised of about 24% Xylose and 2% arabinose, both of which are pentose sugars that are poorly utilized by the industrial yeast strains currently employed (Antoni et al., Appl Microbiol Biotechnol 77:23-35 (2007)).

Several groups have attempted to traverse this problem by genetically engineering strains of Saccharomyces cerevisiae to contain enzymes necessary for efficient metabolism of xylose. In initial attempts, various bacterial xylose isomerases were expressed in S. cerevisiae (Amore et al., Appl Microbiol Biotechnol 30:351-357 (1989); Ho et al., Biotechnol Bioeng Symp 13:245-250 (1983); Moes et al., Biotechnol Lett 18:269-274 (1996); Sarthy et al., Appl Environ Microbiol 53:1996-2000 (1987); Walfridsson, et al., Appl Environ Microbiol 62:4648-51 (1996)). However, only minimal xylose metabolism was found in these recombinant yeast at temperatures suitable for industrial application.

Other groups have tried to enhance the ethanolic fermentation of S. cerevisiae by exogenously expressing P. stipitis xylose reductase (XR) and xylose dehydrogenase (XDH) (Kötter and Ciriacy, Appl Microbiol Biotechnol 38:776-783 (1993); Tantirungkij et al., J Ferm Bioeng 75:83-88 (1993); Walfridsson et al., Appl Microbiol Biotechnol 48:218-224 (1997)). These studies also failed to yield recombinant S. cerevisiae strains that utilized xylose for high yield ethanolic fermentation.

U.S. Pat. No. 7,091,014 to Aristidou et al. describes the genetic engineering of fermenting microorganisms, including S. cerevisiae and Schizosaccharomyces pombe, to express an NAD-dependent glutamate dehydrogenase (GDH) or malic enzyme (ME). These modified yeast display modest increases in ethanol and xylitol production, but do not appear to metabolize xylose any faster than control strains lacking the GDH or ME enzymes.

U.S. Pat. No. 7,253,001 to Wahlbom et al. provides genetically engineered yeast for the ethanolic fermentation of xylose. The engineered yeast of U.S. Pat. No. 7,253,001 recombinantly express exogenous genes for xylose reductase, xylitol dehydrogenase, xylulokinase, phosphoacetyltransferase, aldehyde dehydrogenase, and optionally phosphoketolase.

Similarly, U.S. Pat. No. 7,226,735 to Jeffries and Jin provides genetically engineered yeast strains comprising heterologous gene sequences encoding xylose reductase, xylitol dehydrogenase, and D-xylulokinase enzymes, which are capable of performing fermentation of xylose. U.S. Pat. No. 7,285,403 to Jeffries et al. provides similar engineered yeast strains that additionally display reduced PHO13 expression.

One drawback to using these genetically engineered yeast strains for food and beverage production is that the products, such as ethanol and xylitol, may be regulated as novel GMO (genetically modified organism) produced food. Such regulations may result in additional safety and labeling requirements that are not needed for foods produced by using unmodified organisms. As such, there remains a need in the art for methods of efficiently fermenting pentose and hexose sugars without the use of genetically modified organisms.

Xylitol, a five carbon sugar alcohol, is an increasingly utilized sugar substitute with several desirable properties. First several studies have shown that xylitol provides anticariogenic effects that promote oral health (Tanzer J M., Int Dent J. 1995 February; 45(1 Suppl 1):65-76). Secondly, xylitol metabolism is not regulated by the insulin pathway, which makes this sweetener an attractive sugar substitute for diabetics. Similarly, xylitol is an appropriate sugar substitute for individuals who suffer from glucose-6-phosphate dehydrogenase deficiencies. Finally, xylitol has fewer calories and net effective carbohydrates than does table sugar, making it a viable dietary substitute for sucrose.

Although xylitol is present in many fruits and vegetables, extraction is inefficient and uneconomical. As such, xylitol is industrially produced through the chemical reduction of xylose. Typically, xylan-containing biomass is hydrolyzed to produce a mixture of pentose and hexose sugars, including D-xylose. After enrichment, D-xylose is then converted to xylitol in a chemical process using e.g. a nickel catalyst such as Raney-nickel. Many procedures for this process have been developed, for example see U.S. Pat. Nos. 3,784,408, 4,066,711, 4,075,406, 4,008,285, and 3,586,537. However, the use of xylitol is still limited due to the high costs of production and purification. Accordingly, improved biotechnological processes for the production of xylitol, especially from readily available carbon sources such as corn, sugar cane, and various wood sources high in hemicellulosic biomass, are highly desirable.

Several xylose-metabolizing yeast species have been suggested for use in the production of xylitol, including species of Candida (WO 90/08193, WO 91/10740, WO 88/05467, U.S. Pat. No. 5,998,181), mutant and genetically modified Kluyvermyces (U.S. Pat. No. 6,271,007), Debaryomyces (Rivas et al., Biotechnol Bioeng. 2008 Oct. 3) and genetically modified Saccharomyces (U.S. Pat. No. 7,226,761). However, use of the above yeasts have failed to translate into economically viable industrial procedures for the biotechnological production of xylitol. As such, there remains a need in the art for processes that utilize xylose-metabolizing microorganisms in the industrial production of xylitol.

Nitrogen fixation refers to the biological process by which atmospheric nitrogen (N₂) is converted into ammonia. This process is essential for life because fixed nitrogen is required for the biosynthesis of both amino acids and nucleotides and as such is required for all plant growth. Unfortunately, most plants, including industrially and commercially important crops, are unable to fix nitrogen. These plants rely on nitrogen fixation from various prokaryotes, termed diazotrophs, including species of bacteria and actinobacteria.

Due to the high fixed nitrogen requirements, fixed nitrogen is commonly a limiting resource for plant growth. To combat this, farmers typically rely on fertilizers to supplement the fixed nitrogen content of the soil used for crop growth.

Despite the need for fixed nitrogen supplementation, there are several disadvantages to the use of fertilizers and in particular chemically synthesized inorganic fertilizers. For example, synthesized nitrogen requires high levels of fossil fuels such as natural gas and coal, which are limited resources. In fact, according to the International Fertilizer Industry Association (IFA), production of synthetic ammonia currently consumes nearly 2% of the world energy production with more than 100 million metric tons of ammonia being produced in 2008.

In addition, the run-off of nitrogen-rich compounds found in fertilizers is suspected to be a major contributor to the depletion of oxygen in many parts of the ocean, especially in coastal zones, such as off the coast of the pacific northwestern region of North America. Similarly, methane and nitrous oxide emissions resulting form the use of ammonium based fertilizers may contribute to global climate change, as greenhouse gasses.

Practically speaking, the high cost of growing food crops and biomass for the production of bioenergy (i.e., bioethanol) is in part due to the high cost of fertilizers. As such, methods of nitrogen fixation and crop fertilization that reduce or eliminate the reliance on chemically synthesized fertilizers are needed to reduce the environmental, agricultural, and financial impact that accompany the use of traditional fertilizers.

The present invention provides three novel yeast isolates that are capable of metabolizing a wide range of pentose and hexose sugars, as well as novel methods for the production of bioethanol and xylitol, the fixation of nitrogen, and crop fertilization, which satisfy these and other needs in the art.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides novel endophytic yeast strains capable of metabolizing both pentose and hexose sugars. In a certain embodiment, the yeast strains are selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In specific embodiments, the stains are identified by an rRNA gene sequence selected from any one of SEQ ID NOS:7 to 18.

In a second aspect, the present invention provides biologically pure cultures of the novel endophytic yeast strains of the invention. Cultures of the invention may comprise either a single strain of endophytic yeast or a mixture of microorganisms comprising at least one of the novel yeast strains provided herein.

In another aspect of the invention, methods of producing ethanol are provided. In one embodiment, a method of producing ethanol is provided comprising fermenting a carbon source with an endophytic strain of yeast that is capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18.

In yet another aspect of the invention, methods of producing xylitol are provided. In one embodiment, the method comprises fermenting a carbon source with an endophytic strain of yeast capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18.

In one aspect of the invention, methods of producing mixtures of xylitol and ethanol are provided. In one embodiment, the method comprises fermenting a carbon source with an endophytic strain of yeast capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18.

In another aspect, the present invention provides methods of producing substantially pure ethanol and/or xylitol. In certain embodiments, the methods comprise the steps of producing a mixture of xylitol and ethanol and purifying said xylitol and ethanol from the residual material. In one embodiment, the method comprises fermenting a carbon source with an endophytic strain of yeast capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18.

In yet another aspect, the invention provides a method of producing xylitol, the method comprising the steps of hydrolytically treating a source of biomass, separating a first stream comprising xylose from a second stream comprising glucose, and fermenting said first stream with an endophytic strain of yeast capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18.

In certain embodiments, the method further comprises fermenting said second stream with a yeast capable of producing ethanol. In a particular embodiment, the yeast is an endophytic strain capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18.

In one aspect, the invention provides methods of producing animal feedstock. In certain embodiments, the methods comprise fermenting a carbon source. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18.

In another aspect, the invention provides a recombinant yeast capable fermenting both hexose and pentose sugars. In certain embodiments, the yeast harbors a heterologous gene sequence from an endophytic yeast strain. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18. In certain embodiments, the recombinant yeast is a Saccharomyces or a Schizosaccharomyces yeast strain.

In yet another aspect, the invention provides a method of producing ethanol. In one embodiment, the method comprises fermenting a carbon source with a recombinant yeast capable of fermenting both pentose and hexose sugars. In certain embodiments, the yeast comprises a heterologous gene sequence from an endophytic yeast strain capable of fermenting both pentose and hexose sugars. In certain embodiments, the yeast comprises a heterologous gene sequence from an endophytic yeast strain capable of fermenting both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18. In certain embodiments, the recombinant yeast is a Saccharomyces or a Schizosaccharomyces yeast strain.

In one aspect, the invention provides novel Xylose Dehydrogenase (XDH) and Xylose Reductase (XR) genes and coding sequences cloned from the endophytic yeast provided herein, as well as the polypeptides encoded therein.

In another aspect, the invention provides a method of fixing nitrogen comprising the use an endophytic yeast of the invention or a recombinant organism harboring a heterologous gene from an endophytic yeast provided herein. In certain embodiments, the method comprises fertilization of a crop.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Photomicrographs of the yeast strains (scale bar equal to 25 mm for A and 10 mm for B, C, D). (A) WP1; (B) PTD2; (C) PTD3; (D) Rhodotorula glutinis.

FIG. 2. Phylogenetic tree showing relatedness among 18S gene sequences of yeast strains. The tree was constructed with a total of 952 positions using a neighbor joining distance matrix. Evolutionary distances were computed using the Jukes-Cantor method. Bootstrap values (1000 tree interactions) are indicated at the nodes.

FIG. 3. Phylogenetic reconstruction based on ITS1-5.8S-ITS2 sequences of yeast strains. The tree was constructed with a total of 583 positions using a neighbor-joining distance matrix. Evolutionary distances were computed using the Jukes-Cantor method. Bootstrap values (1000 tree interactions) are indicated at the nodes.

FIG. 4. Phylogenetic tree showing relatedness among large subunit gene D1/D2 region sequences of yeast strains. The tree was constructed with a total of 586 positions using a neighbor-joining distance matrix. Evolutionary distances were computed using the Jukes-Cantor method. Bootstrap values (1000 tree interactions) are indicated at the nodes.

FIG. 5. Phylogenetic tree showing relatedness among 18S gene sequences of yeast strains. The tree was constructed with a total of 952 positions using a Maximum Parsimony method. Bootstrap values (1000 tree interactions) are indicated at the nodes.

FIG. 6. Phylogenetic reconstruction based on ITS1-5.8S-ITS2 sequences of yeast strains. The tree was constructed with a total of 583 positions using a Maximum Parsimony. Bootstrap values (1000 tree interactions) are indicated at the nodes.

FIG. 7. Phylogenetic tree showing relatedness among large subunit gene D1/D2 region sequences of yeast strains. The tree was constructed with a total of 586 positions using a Maximum Parsimony method. Bootstrap values (1000 tree interactions) are indicated at the nodes.

FIG. 8. Clustering of phenotypic characteristics profiles of the studied yeast strains (WP1, PTD2, PTD3, ATCC, and Baker's yeast) and the reference species of the API 20C AUX system (bioMerieux, 2007) based on the overall similarity. The distance between any two clusters was determined by the Ward's minimum variance method (Milligan, 1980).

FIG. 9. IAA production by yeast strains incubated with 0.1% L-tryptophan.

FIG. 10. Growth rates of the yeast strains WP1, PTD2, PTD3, ATCC, and Baker's yeast in rich medium.

FIG. 11. Experimental growth curves for the yeast strains WP1, PTD2, PTD3, ATCC, and Baker's yeast in YPG medium.

FIG. 12. Glucose consumption, glycerol production, and ethanol production in a culture PTD3 grown in glucose.

FIG. 13. HPLC chromatograms for the data provided in FIG. 15, showing the amount of the xylose peak at the beginning of the experiment.

FIG. 14. HPLC chromatograms for the data provided in FIG. 15, showing the reduction of the xylose peak and formation of the xylitol peak at the end of the experiment.

FIG. 15. Consumption of glucose and xylose and production of xylitol and ethanol in a culture of PTD3 yeast grown in glucose and xylose separately.

FIG. 16. Conversion of xylose to xylitol by the yeast strains WP1, PTD2, and PTD3 cultured in xylose.

FIG. 17. Consumption of glucose and xylose and production of xylitol and ethanol in a culture of WP1 yeast grown in glucose and xylose separately.

FIG. 18. Consumption of glucose and xylose and production of xylitol and ethanol in a culture of PTD2 yeast grown in glucose and xylose separately.

FIG. 19. Consumption of glucose and xylose and production of xylitol and ethanol in a culture of PTD3 yeast grown in glucose and xylose separately.

FIG. 20. Effect of medium conditions on the consumption of glucose and the production of ethanol and xylitol by PTD3 yeast.

FIG. 21. Effect of medium conditions on the consumption of xylose and the production of ethanol and xylitol by PTD3 yeast.

FIG. 22. Effect of yeast concentration on the consumption of glucose and the production of ethanol and xylitol by PTD3 yeast.

FIG. 23. Effect of yeast concentration on the consumption of xylose and the production of ethanol and xylitol by PTD3 yeast.

FIG. 24. Fermentation of glucose to ethanol by PTD3 yeast cultured in glucose with MS and yeast extract.

FIG. 25. Fermentation of xylose to xylitol by PTD3 yeast cultured in xylose with MS and yeast extract.

FIG. 26. Double fermentation of glucose and xylitol to ethanol and xylitol by PTD3 yeast cultured in glucose and xylose together and MS and yeast extract.

FIG. 27. Growth rate of PTD3 yeast in medium containing glucose and xylose.

FIG. 28. Mixed fermentation of hexose (28A) and pentose (28B) sugars by PTD3 yeast cultured in MS and yeast extract with arabinose, galactose, glucose, xylose and mannose.

FIG. 29. Growth rate of PTD3 yeast grown in mediums containing mixed sugars, arabinose, xylose, glucose, galactose, and mannose with MS and yeast extract.

FIG. 30. Growth rate of PTD3 yeast grown in mediums containing mixed sugars, arabinose, xylose, glucose, galactose, and mannose with MS and yeast extract.

FIG. 31. Mixed fermentation of glucose and xylitol to ethanol and xylitol by PTD3 yeast cultured in arabinose, xylose, glucose, galactose, and mannose with MS and yeast extract.

FIG. 32. 18S Ribosomal sequence (SEQ ID NO:18) for the Ad1 yeast isolated from Arundo donax (giant reed).

FIG. 33. Growth curve of Rhodotorula graminis strain WP1 and Saccharomyces cerevisiae (ATCC strain #6037) in nitrogen-free MS medium (Caisson) containing dextrose and mannitol. Growth in three flasks of each strain was monitored for 3 days. The experiment was repeated with similar results.

FIG. 34. (A) Growth of WP1 and PTD3 in MS medium with 3% glucose as the carbon source. Baker's yeast (BK; ATCC6037) was used as a positive control. The experiments were performed in triplicate and the error bars indicate the standard deviations. (B) Growth of WP1 and PTD3 in MS medium with 3% xylose as the carbon source. Baker's yeast (BK) was used as a control. The experiments were performed in triplicate and the error bars indicate the standard deviations.

FIG. 35. Exon/Intron structures of the XR and XDH-encoding genes of Rhodotorula graminis strain WP1.

FIG. 36. Exon/Intron structures of the XR and XDH-encoding genes of Pichia stipitis.

FIG. 37. Amplification of WP1 XR mRNA (A) and XDH mRNA (B) from cells grown in glucose or xylose. The first lane is a Fermentas 1 kb DNA ladder. RNA templates directly subjected to a regular PCR (without reverse transcriptase) served as negative controls for both genes. The 1 kb bands were cloned and the sequences were verified to be XR and XDH-encoding genes.

FIG. 38. Amplification of PTD3 XDH mRNA and XR mRNA from cells grown in glucose (“glu”) or xylose (“xyl”). The first lane is a Fermentas 100 bp DNA ladder.

FIG. 39. WP1 and PTD3 XR/XDH gene expressions from cells grown in 2% glucose (lanes 1, 4, 7, 10), 1% glucose+1% xylose (lane 2, 5, 8, 11), and 2% xylose (lanes 3, 6, 9, 12). Lane 1-6: WP1 gene expression; Lane 7-12: PTD3 gene expression.

FIG. 40. WP1 and PTD3 XR (right)/XDH (left) gene expressions from cells grown in 2% glucose, 2% xylose and 2% glucose+2% xylose. RNA templates used in different conditions were labeled in the figure.

FIG. 41. WP1 and PTD3 XR/XDH gene 18S rRNA RT-PCR from cells grown in 2% glucose (lanes 1, 3), 2% glucose+2% xylose (lanes 2, 4) and 2% xylose (lanes 3, 6) medium. Lane S is a Fermentas 1 kb DNA ladder.

FIG. 42. Corn growth after 11 weeks in nitrogen-limited conditions with or without WP1 inoculation. Three different corn varieties (lines 1, 2, and 3) were planted in each container. Biomass of the uninoculated plants was 9.3 g, 3.9 g, and 15.0 g whereas the biomass of the WP1 inoculated plants was 63 g, 87.1 g, and 45.1 g. In addition, the % viability in WP1 colonized plants (58-92%) was higher than uninoculated plants (8.3-29.2%) plants. Statistical analysis indicated significant differences (P≦0.1) for both viability and biomass with WP1 symbiotic plants having higher viability and biomass compared to uninoculated plants.

FIG. 43. Greenhouse studies with corn (line-3) that were either NS=nonsymbiotic (uninoculated control) or symbiotic (N=9) with Rhodotorula sp. WP1 in the absence of stress. The biomass, yields and heights of plants were assessed, and WP1 colonized plants were found to be larger and produced higher yields (ears) than NS plants. (P≦0.004). N=9; SE≦0.21, 0.56, and 0.27 for biomass, yields, and heights, respectively.

FIG. 44. PTD3 XDH-encoding gene open reading frame (SEQ ID NO:47).

FIG. 45. PTD3 XR-encoding gene open reading frame (SEQ ID NO:45).

FIG. 46. WP1 XR-encoding gene open reading frame (SEQ ID NO:41).

FIG. 47. WP1 XDH-encoding gene open reading frame (SEQ ID NO:43).

DETAILED DESCRIPTION OF THE INVENTION I. Overview

In one aspect, the present invention provides novel endophytic yeast strains, including WP1 and PTD3, isolated from within the stems of poplar (Populus) trees, which were genetically characterized with respect to their xylose metabolism genes. These strains, belonging to species Rhodotorula graminis and R. mucilaginosa, respectively, utilize both hexose and pentose sugars, including the common plant pentose sugar, D-xylose. In another aspect, the present invention provides the xylose reductase gene (XYL1) and xylitol dehydrogenase gene (XYL2) from these yeast strains, which were cloned and characterized. The derived amino acid sequences of xylose reductase (XR) and xylose dehydrogenase (XDH) are 32°˜41% homologous to those of Pichia stipitis and Candida. Spp., species known to utilize xylose. The derived XR and XDH sequences of WP1 and PTD3 have higher homology (73% and 69% identity) with each other. WP1 and PTD3 were grown in single sugar and mixed sugar medium to analyze the XYL1 and XYL2 gene regulation mechanisms. These results revealed that for both strains, the gene expression is induced by D-xylose, and that the expression was not repressed by glucose in the presence of xylose in PTD3.

Notably, the gene expression of the WP1 and PTD3 is unique in these endophytic yeast strains. They are expressed in response to xylose even when glucose is present. In contrast, in other species, these xylose metabolism genes are shut off when glucose is present, preventing simultaneous use of both 5-carbon and 6-carbon sugars.

Lignocellulosic material containing cellulose, hemicellulose, and lignin is an abundant renewable organic resource that can be used for the production of energy and biochemicals. The conversion of both the cellulose and hemicellulose fractions for production of biochemicals is being studied intensively. Between 23% to 40% of the lignocellulosic biomass consists of hemicellulose, the main component being xylose in most hardwoods and annual plants (Lee et al. 1979). Whereas the fermentation of glucose can be carried out efficiently by the common brewer's yeast (Saccharomyces cerevisiae), the bioconversion of the pentose fraction (xylose and arabinose) presents a challenge since it is not metabolized by this species. In the past decades, numerous studies have been carried out on various aspects of D-xylose bioconversion (Du Preez 1994; Winkelhausen and Kuzmanova 1998).

D-xylose can be utilized by bacteria, yeasts and fungi (Jeffries 1983) using different pathways. In one pathway, D-xylose can be directly converted to D-xylulose by xylose isomerase (Aristidou and Penttila 2000) without the participation of cofactors. In some yeasts and fungi, conversion of D-xylose to D-xylulose is carried out more often by two enzymatic steps. First, D-xylose is reduced by a NADPH/NADH-linked xylose reductase (XR) to xylitol, followed by oxidation of xylitol to xylulose by an NAD-linked xylitol dehydrogenase (XDH) (Bruinenberg and van Dijken 1983). D-xylulose is subsequently phosphorylated to D-xylulose-5-phosphate by D-xylulokinase before it enters the pentose phosphate, Embden-Meyerhof, and phosphoketolase pathways (Skoog and Hahn-Hagerdal 1988).

The two major chemicals of interest that can be produced from D-xylose by yeasts are ethanol and xylitol. It is known that under normal growth conditions, some pentose-fermenting yeasts (e.g. Pichia stipitis) produce mostly ethanol (Du Preez 1994; Schneider 1989); while others (e.g. Candida guilliermondii, Candida tropicalis) produce mainly xylitol as the end products (Barbosa et al. 1988; Gong et al. 1981). As an intermediate metabolite, xylitol is widely applied in food and pharmaceutical industries because of its equivalent sweetness to sucrose and high negative heat of solution (Borges 1991; Passon 1993), its anti-cariogenic and anti-infection effects (Pizzo et al. 2000; Sakai et al. 1996; Brown et al. 2004), and independent metabolism of insulin, therefore making it useful for diabetic patients (Salminen et al. 1989). Among the xylose-fermenting yeast, the genus Candida is one of the most efficient xylitol producers (Meyrial et al. 1991). Ojama demonstrated that C. guilliermondii VTT-C-71006 is an efficient xylitol producer. A xylitol yield of 0.74 g/g xylose was obtained within 50 hours at an initial D-xylose concentration of 100 g/l (Ojama 1994).

The pink yeast strains WP1 (Rhodotorula graminis) and PTD3 (Rhodotorula mucilaginosa) provided in one aspect of the present invention are remarkable for their good performance in xylitol production (approximately 67% conversion) and sugar metabolism in the presence of several common fermentation inhibitors (Vajzovic, A., unpublished). So far, investigation of xylitol production by yeasts has been limited to Candida and Pichia species and studies of D-xylose metabolism in Rhodotorula. spp were barely reported. Although XR and XDH activities were detected in Rhodosporidium toruloides (the teleomorph of Rhodotorula glutinis) (Freer et al. 1997), none of the genes encoding XR and XDH were cloned from the Rhodotorula genus. The present invention provides, among other aspects, the first report that describes the cloning and characterization of the XR-encoding gene (XYL1) and XDH-encoding gene (XYL2) from both Rhodotorula graminis and Rhodotorula mucilaginosa yeast strains.

In one aspect, the present invention provides XR and XDH encoding genes, which were cloned from Rhodotorula graminis strain WP1. The expression of the two genes was verified by RT-PCR. This study shows that D-xylose is a good inducer of XR and XDH in both strains. This is similar to the trend found with Candida guilliermondii (Sugai and Delgenes 1995) and Pichia stipitis (Bichio et al. 1988). Furthermore, a novel characteristic of lack of inhibition by glucose for these genes is also demonstrated.

Notably, the XI (xylose isomerase)-encoding gene was not found in the WP1 genome sequence provided by the JGI. Thus WP1 likely utilizes the two-step redox pathway in D-xylose metabolism as in other yeasts. However, the alignments showed that the XR and XDH sequences have low homology (32%˜41% identities) with other XRs and XDHs from Candida spp. and Pichia stipitis yeasts. In addition, the WP1 XR and XDH-encoding genes have multiple introns and the exon/intron structures are more complicated and advanced than the homologous genes in Pichia stipitis and Candida spp. These differences might introduce greater variability of protein sequences translated from a single gene and might have an impact on enhancing the expression of the XR and XDH genes (Smith and Lee 2008; Lin et al. 2010). From the macro perspective, the gene differences suggest that there could be long evolution distances between WP1 and Pichia stipitis and Candida. spp and this might lead to some other differences in the xylose metabolism pathway between these yeasts.

The present study of gene expression levels in xylose and glucose media shows that the expression of WP1 XR and XDH-encoding genes were induced by xylose. The two genes in WP1 were expressed to low levels while grown in glucose medium. Additionally, the expression level of the XDH-encoding gene (XYL1) in WP1 was higher than that of the XR-encoding gene (XYL2).

In a related aspect, the present invention also provides full-length XR and XDH encoding genes, which were cloned from Rhodotorula mucilaginosa strain PTD3. The expression of these two genes was also verified by RT-PCR. Sequence alignment results show that the XR and XDH sequences also have low homology (37%˜41% identities) with other XRs and XDHs from Candida spp. and Pichia stipitis yeasts. Since the genome sequence of PTD3 is not available, the exon/intron structures of the two genes was not determined. However, based on the high homology (73% and 69% identity for XR, XDH) with WP1, it is likely that the gene structures of PTD3 may be more like that of WP1 and that these two endophytic yeasts of poplar trees may metabolize D-xylose using the same pathway.

Like in WP1, the expression of PTD3 XR and XDH-encoding genes was also induced by xylose. The two genes in PTD3 were expressed to low levels while grown in glucose medium. As in WP1, the expression level of the XDH-encoding gene (XYL1) in PTD3 was higher than that of the XR-encoding gene (XYL2).

Since PTD3 grew better in D-xylose medium compared to WP1, one hypothesis to explain this difference is that PTD3 may produce the enzymes involved in xylose metabolism at higher levels than does WP1. This gene expression study verified that both the XR and XDH-encoding gene expression levels were much higher in PTD3 than in WP1, thus supporting this hypothesis. Further study into the resulting protein levels and also the xylose uptake mechanisms for these yeast strains is yet to be explored.

In another aspect of the invention, single sugars and mixed sugars were investigated to analyze their potential to induce XR and XDH-encoding gene expressions in both WP1 and PTD3. For many yeasts like Saccharomyces cerevisiae, Pichia stipitis and Candida. spp, D-glucose is the preferred substrate for growth and fermentation when both D-glucose and D-xylose are present in the medium. The genes for xylose assimilation (XYL1, XYL2) were not expressed in Pichia stipitis in the presence of glucose in the medium (Jeffries et al. 2007). The present study shows that in both WP1 and PTD3 yeast strains, the two genes were still expressed in response to xylose in the presence of glucose in the medium. Furthermore, the band quantities of RT-PCR in single sugar (xylose) and mixed sugar (glucose+xylose) revealed that the two genes were not repressed by glucose in PTD3 while they were slightly suppressed in WP1. These are significant results because xylose reductase and xylitol dehydrogenase are pivotal for growth and xylitol formation during xylose metabolism. And the high-level expression of both genes in the mixed sugars of xylose and glucose will largely increase the xylitol yield in mixed sugars from real hydrolytes and will contribute to optimizing fermentation conditions of lignocellulosic biomass. In addition, better understanding of the regulation mechanism of these genes, together with identification of the XR and XDH-encoding genes as well as the xylose uptake genes will help determine the strategies for genetic engineering of industry strains such as S. cerevisiae for further improvement of productivity. Accordingly, the present invention provides, in one aspect, recombinant yeast cells and strains harboring a heterologous XR and/or XDH-encoding gene from the WP1 or PTD3 strain, or a highly similar sequence.

In one aspect, the present invention provides a biotechnological process for the production of a sugar alcohol or polyol, using the endophytic yeast provided herein. One novel yeast strain, provided herein, was isolated from poplar trees and has several unique properties. In one embodiment, the invention provides a PTD3 yeast strain isolated from a hybrid poplar tree or a giant reed.

Pretreatment of lignocellulosic biomass can produce fermentation inhibitors such as furfural, 5-HMF, and acetic acid. These compounds can decrease the ethanol yields from sugars. Provided herein are isolated yeast strains that have a high tolerance for such inhibitors. A systematic study of the effect of furfural, 5-HMF, and acetic acid concentration on the fermentation of glucose and xylose to ethanol and xylitol respectively by PTD3, a novel, genetically unmodified yeast is provided herein.

The influence of furfural in different concentrations (from 1 to 5 g/L) on the growth of PTD3 yeast under cultivation in synthetic nutrient media has been studied. The yeast provided herein grow well in presence of furfural and showed resemblance in growth and fermentative pattern with controls. Ethanol yield achieved from glucose and xylitol, using the yeast strains of the invention, were of 90% of theoretical yield for ethanol and 70% of the theoretical yield for xylitol. Ethanol yields from glucose were not influenced by presence of furfural. However, xylitol biosynthesis was affected by the presence of furfural in the fermentation media. The effects of higher concentrations of furfural (10 and 20 g/L) on the ethanol and xylitol yields are presented herein, as well as the effects of 5-HMF and acetic acid.

Up to date, there is no reported microorganism that is capable of utilizing both, hexose and pentose sugars at the same time, without being genetically modified or co-cultured. A genetically unmodified yeast which is capable of rapid assimilation and catabolism of five and six carbon sugars (arabinose, xylose, galactose, glucose and mannose) is provided herein. This yeast (PTD3) was shown not to be subject to hexose-mediated repression during mixed sugars fermentation. PTD3 produced ethanol of 82% of theoretical during fermentation of glucose, mannose and galactose. It produced considerable amount of xylitol of 96.1% of theoretical when xylose was present in the fermentation media. The high ethanol and xylitol were obtained without media, aeration, temperature and pH optimization.

The novel yeast provided herein also have a high tolerance of inhibitors, including without limitation, furfurals, 5-HMF, and acetic acid, during biological production of ethanol and xylitol. PTD3 can effectively ferment five and six carbon sugars present in hydrolysates from different cellulosic biomass, for example, steam pretreated switchgrass, hybrid poplar, and sugar cane bagasse, to ethanol and xylitol.

II. Endophytic Yeast Strains and Cultures Thereof

In one embodiment of the invention, novel endophytic yeast strains capable of metabolizing both pentose and hexose sugars are provided. In certain embodiments, these yeast strains are most closely related to Rhodotorula graminis or Rhodotorula mucilaginosa species. In a particular embodiment, the novel strains of the invention are selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In one embodiment of the invention, the novel endophytic yeast strains contain an rRNA gene sequence that is selected from any one of SEQ ID NOS:7 to 18. In a particular embodiment, an endophytic yeast strain of the invention may have an 18S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to 18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

In another embodiment, the present invention provides cultures of novel endophytic yeast strains capable of metabolizing both pentose and hexose sugars. In some embodiments, the cultures of the invention comprise a biologically pure culture of an endophytic yeast strain, while in other embodiments, the cultures of the invention may comprise more than one strain of yeast. In specific embodiments, the cultures of the invention may comprise a yeast strain selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the cultures of the invention comprise one or more yeast strain that is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18.

The novel endophytic yeast strains and cultures of the present invention may be useful in the fermentative production of bioethanol, xylitol, and other biotechnological manufacturing products. In a particular embodiment, the novel strains of the invention are useful for the fermentation of mixtures of pentose and hexose sugars. In certain embodiments, the strains and cultures of the invention are useful for the fermentation of biomass that has been pretreated to yield a mixture of pentose and hexose sugars. For example, lignocellulosic biomass such as wood or wood residuals (e.g., saw mill or paper mill discards), municipal paper waste (e.g., newspapers), agricultural residuals (e.g., corn stover, sugarcane bagasse), tall woody grasses, and the like. Methods of pretreating lignocellulosic biomass for yeast fermentation are well known in the art and include both acid hydrolysis and enzymatic hydrolysis. For review, see Lange J. P., Biofuels, Bioproducts, and Biorefining 1(1):39-48 (2007); Jørgensen H. et al., Biofuels, Bioproducts, and Biorefining 1(2):119-134 (2007); Wyman C. E. et al., Bioresour Technol. 2005 December; 96(18):2026-32; and Wyman C. E. et al., Bioresour Technol. 2005 December; 96(18):1959-66.

In another embodiment, the endophytic yeast strains and cultures of the present invention may be useful for fixing atmospheric nitrogen. In a particular embodiment, the novel strains of the invention are useful for fertilizing a crop in the presence or absence of a traditional chemical fertilizer. In one embodiment, the novel strains are useful for inoculating a crop or colonizing the soil a crop is planted in with the yeast. The soil may be colonized with the yeast prior to planting the crop, for example before, during, or after tilling the soil in preparation for planning the crop. In other embodiments, the soil may be colonized with the yeast after the crop has been planted.

In some embodiments, the cultures of the invention useful for nitrogen fixation and/or fertilization of a crop comprise a biologically pure culture of an endophytic yeast strain, while in other embodiments, the cultures of the invention may comprise more than one strain of yeast. In specific embodiments, the cultures of the invention may comprise a yeast strain selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the cultures of the invention comprise one or more yeast strain that is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18.

III. Methods for Producing Ethanol, Sugar Alcohols, and Polyols

In one embodiment, the present invention provides novel methods for producing ethanol comprising fermenting a carbon source with an endophytic strain of yeast. In certain embodiments, the endophytic yeast is capable of metabolizing both pentose and hexose sugars. In a specific embodiment, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18. In a particular embodiment, an endophytic yeast strain of the invention may have an 18S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to 18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

In another embodiment of the invention, methods of producing xylitol are provided. In one embodiment, the method comprises fermenting a carbon source with an endophytic strain of yeast capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18. In a particular embodiment, an endophytic yeast strain of the invention may have an 18S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to 18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

The carbon sources used in the methods of the invention, may comprise a pentose sugar or sugar alcohol, a hexose sugar or sugar alcohol, or a combination thereof. In particular embodiments, the carbon source is selected from the group consisting of glucose, glycerol, calcium 2-keto-gluconate, arabinose, xylose, adonitol, xylitol, galactose, inositol, sorbitol, methyl-α-glucopyranoside, N-acetyl-glucosamine, cellobiose, lactose, maltose, sucrose, trehalose, melezitose, raffinose, and combinations thereof. In a particular embodiment, the carbon source is xylitol, glucose, or a combination of sugars containing xylitol, glucose, or both. In other embodiments, the carbon source may comprises biomass that has been hydrolytically pre-treated. For example, lignocellulosic biomass such as wood or wood residuals (saw mill or paper mill discards), municipal paper waste, agricultural residuals (corn stover, sugarcane bagasse), tall woody grasses, and the like. The carbon sources used in the methods of the invention are not limited to those listed above.

In related embodiments, the present invention provides methods of producing mixtures of xylitol and ethanol. In one embodiment, the method comprises fermenting a carbon source with an endophytic strain of yeast capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18. In a particular embodiment, an endophytic yeast strain of the invention may have an 18S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to 18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15. In one particular embodiment, the present invention provides methods of producing mixtures of xylitol and ethanol.

In certain embodiments, the methods of the invention further comprise purifying one or more of xylitol, ethanol, or both after production. Methods of purifying xylitol from reaction mixtures are well known in the art and include, without limitation, distillation, crystallization, chromatography, combinations thereof, and the like. For example, U.S. Pat. No. 6,538,133 describes chromatographic procedures of purifying xylitol from cultures of xylitol-producing microorganisms. Rivas et al., J. Agric. Food Chem. 2006, 54(12):4430-4435, describe a process of purifying xylitol obtained by fermentation of corncob hydrolysates by crystallization. Methods of distilling ethanol are also well known in the art. For example, U.S. Pat. No. 7,297,236 describe process arrangements for distilling fuel grade ethanol. Methods of simultaneously producing xylitol and ethanol are well known in the art, for example see U.S. Pat. No. 7,109,055.

In certain embodiments, the methods of the present invention comprise the steps of producing a mixture of xylitol and ethanol and purifying said ethanol and xylitol from the residual material. In one particular embodiment, the mixture of xylitol and ethanol is first distilled to yield substantially pure ethanol and then xylitol is purified from the distillation residuals. In one embodiment, the method comprises fermenting a carbon source with an endophytic strain of yeast capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18. In a particular embodiment, an endophytic yeast strain of the invention may have an 18S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to 18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

In yet other embodiments of the invention, methods are provided for the production of xylitol comprising the steps of hydrolytically treating a source of biomass to produce a mixture of pentose and hexose sugars, separating a first stream comprising xylose from a second stream comprising glucose, and fermenting said first stream with an endophytic strain of yeast capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18. In a particular embodiment, an endophytic yeast strain of the invention may have an 18S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 15 to 16, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

In some embodiments, the above method further comprises fermenting said second stream with a yeast capable of producing ethanol. In a particular embodiment, the yeast is an endophytic strain capable of metabolizing both pentose and hexose sugars. In certain embodiments, the endophytic strain is selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In other embodiments, the endophytic strain is identified by an rRNA gene sequence selected from the group consisting of SEQ ID NOS:7 to 18. In a particular embodiment, an endophytic yeast strain of the invention may have an 18S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to 18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15. In some embodiments, the yeast strain used to ferment said first stream is the same as the yeast strain used to ferment said second stream. In yet other embodiments, the yeast strains are different.

IV. Recombinant Yeast Strains and Methods of Use Thereof

In another aspect, the invention provides recombinant yeast strains capable of fermenting both pentose and hexose sugars. In certain embodiments, these strains harbor a heterologous gene sequence from an endophytic yeast strain selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1, wherein said strain is identified by an rRNA gene sequence selected from any one of SEQ ID NOS:7 to 18.

In one embodiment, the heterologous gene sequence encodes for a xylose reductase (XR) protein or a xylose dehydrogenase (XDH) protein. In certain embodiments, the heterologous gene sequence has at least 85% sequence identity with an XYL1 or XYL2 gene sequence or coding sequence from an endophytic yeast provided herein. In certain embodiments, the heterologous gene sequence has at least 85% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47. In other embodiments, the heterologous gene sequence may have at least about 85% identity, or at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an XYL1 or XYL2 gene sequence or coding sequence provided herein. In certain embodiments, the heterologous gene sequence may further comprise one or more introns.

In one embodiment, the heterologous gene sequence encodes for a xylose reductase (XR) protein or a xylose dehydrogenase (XDH) protein. In certain embodiments, the xylose reductase protein is from the WP1 or the PTD3 stain. In a particular embodiment, the heterologous gene sequence encodes for a polypeptide having at least about 85%, or at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an amino acid sequence selected form SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.

In certain embodiments, the heterologous gene sequence may be cloned into a microbial genome, for example a bacterial or yeast chromosome, or may comprise an expression vector, a recombinant or artificial microbial chromosome, for example a bacterial (BAC) or yeast (YAC) chromosome, a bacterial plasmid, a yeast plasmid, a recombinant bacteria phage, a recombinant viral vector, a mammalian expression vector, a baculovirus vector. In yet other embodiments, the heterologous gene sequence may encode for a fusion protein. In another embodiment, the heterologous gene sequence may encode for a tagged protein, such as a tagged XR or XDH protein.

In certain embodiments, the recombinant yeast strains may be a Saccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, a Brettanomyces, a Torulaspora, an Ascobotryozyma, a Citeromyces, a Debaryomyces, an Eremothecium, a Issatchenkia, a Kazachstania, a Kluyveromyces, a Kodamaea, a Kregervanrija, a Kuraishia, a Lachancea, a Lodderomyces, a Nakaseomyces, a Pachysolen, a Pichia, a Saturnispora, a Tetrapisispora, a Torulaspora, a Vanderwaltozyma, a Williopsis, and the like. In a particular embodiment, the recombinant yeast is a Saccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, or a Brettanomyces. In one embodiment, the strain is Saccharomyces cerevisiae. In a related aspect, biologically pure cultures of the recombinant yeast strains are provided.

In another aspect, the invention provides methods of fermenting a carbon source with a recombinant yeast strain provided herein. In certain embodiments, the methods comprise culturing a recombinant yeast of the invention in the absence of a supplemental nitrogen source, for example ammonium or nitrate.

In a related aspect, methods of producing ethanol are provided. In certain embodiments, the methods comprise fermenting a carbon source with a recombinant yeast strain provided herein. In certain embodiments, the methods comprise fermenting a carbon source in the absence of a supplemental nitrogen source, for example ammonium or nitrate.

In another related embodiment, methods of producing an animal feedstock are provided. In certain embodiments, the method comprises culturing a recombinant yeast of the invention in the absence of a supplemental nitrogen source, for example ammonium or nitrate. Advantageously, these methods provide an inexpensive source of animal feedstock, as the recombinant yeast provided herein are capable of performing nitrogen fixation and thus can be grown in culture medium that is not supplemented with a nitrogen source

The yeast of the invention may be genetically modified to further enhance the metabolism of particular pentose and or hexose sugars. Exogenous genes encoding for any one of a number of enzymes may be introduced and expressed in an endophytic yeast used in any one of the methods of the invention. Non-limiting examples of exogenous enzymes that may be expressed in the yeast of the invention include, xylose isomerases, xylose reductases, xylose dehydrogenases, NAD-dependent glutamate dehydrogenases, malic enzymes, xylulokinases, phosphoacetyltransferase, aldehyde dehydrogenase, phosphoketolase, and the like. Examples of the metabolic engineering of yeasts can be found, for example, in Nevoigt, Microbiology and Molecular Biology Reviews 2008 72(3):379-412.

In certain embodiments, the methods of the invention comprise optimizing the culture medium in order to maximize the production of a particular product, such as ethanol or xylitol.

In another embodiment, the recombinant yeast strains and cultures provided herein may be useful for fixing atmospheric nitrogen. In a particular embodiment, the novel strains are useful for fertilizing a crop in the presence or absence of a traditional chemical fertilizer. In one embodiment, the novel strains are useful for inoculating a crop or colonizing the soil a crop is planted in with the yeast. The soil may be colonized with the yeast prior to planting the crop, for example before, during, or after tilling the soil in preparation for planning the crop. In other embodiments, the soil may be colonized with the yeast after the crop has been planted.

V. Xylose Reductase and Xylose Dehydrogenase Polynucleotides and Polypeptides

In one aspect, the present invention provides xylose reductase (XR) polypeptides and xylose dehydrogenase (XDH) polypeptides. In certain embodiments, XR and XDH polypeptides are from a yeast strain selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In some embodiments, the strain is identified by an rRNA gene sequence selected from any one of SEQ ID NOS:7 to 18. In one embodiment, the polypeptide has an amino acid sequence that is at least about 85% identical to a an amino acid sequence selected form SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48. In other embodiments, the amino acid sequence of the protein has at least about 85%, or at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an amino acid sequence selected form SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48. In one embodiment, the invention provides a polypeptide encoded by the nucleotide sequence found in FIG. 44, FIG. 45, FIG. 46, or FIG. 47. In other embodiments, the amino acid sequence of the protein has at least about 85%, or at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an amino acid sequence encoded by the nucleotide sequence found in FIG. 44, FIG. 45, FIG. 46, or FIG. 47.

In a related aspect, the present invention provides isolated and/or recombinant polynucleotides encoding for a xylose reductase (XR) polypeptide and/or a xylose dehydrogenase (XDH) polypeptide. In certain embodiments, XR and XDH polypeptides are from a yeast strain selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In some embodiments, the strain is identified by an rRNA gene sequence selected from any one of SEQ ID NOS:7 to 18. In one embodiment, the polypeptide encoded by a polynucleotide of the invention has an amino acid sequence that is at least about 85% identical to a an amino acid sequence selected form SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48. In other embodiments, the amino acid sequence of a polypeptide encoded by a polynucleotide of the invention has at least about 85%, or at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an amino acid sequence selected form SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48. In one embodiment, the invention provides a polynucleotide that encodes for a polypeptide encoded by the nucleotide sequence found in FIG. 44, FIG. 45, FIG. 46, or FIG. 47. In other embodiments, the invention provides a polynucleotide that encodes for a polypeptide with an amino acid sequence that has at least about 85%, or at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an amino acid sequence encoded by the nucleotide sequence found in FIG. 44, FIG. 45, FIG. 46, or FIG. 47.

In a related embodiment, the present invention provides isolated and/or recombinant polynucleotides comprising an XYL1 and/or XYL2 gene or coding sequence from an endophytic yeast provided herein. In certain embodiments, the polynucleotide comprises a nucleotide sequence that has at least 85% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47. In other embodiments, the polynucleotide comprises a nucleotide sequence that has at least about 85% identity, or at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an XYL1 or XYL2 gene sequence or coding sequence provided herein, for example, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:47. In certain embodiments, the polynucleotide may further comprise an intron or intronic sequence. In one embodiment, the polynucleotide comprising an XYL1 and/or XYL2 gene or coding sequence comprises a nucleotide sequence found in FIG. 44, FIG. 45, FIG. 46, or FIG. 47. In certain embodiments, the polynucleotides of the present invention may further comprise one or more introns.

In one embodiment, the polynucleotide sequence encodes for a xylose reductase (XR) protein or a xylose dehydrogenase (XDH) protein. In certain embodiments, the xylose reductase protein is from the WP1 or the PTD3 stain. In a particular embodiment, the heterologous gene sequence encodes for a polypeptide having at least about 85%, or at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to an amino acid sequence selected form SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.

In certain embodiments, the polynucleotide may comprise a microbial genome, for example a bacterial or yeast chromosome, or may comprise an expression vector, a recombinant or artificial microbial chromosome, for example a bacterial (BAC) or yeast (YAC) chromosome, a bacterial plasmid, a yeast plasmid, a recombinant bacteria phage, a recombinant viral vector, a mammalian expression vector, a baculovirus vector. In yet other embodiments, the heterologous gene sequence may encode for a fusion protein. In another embodiment, the heterologous gene sequence may encode for a tagged protein, such as a tagged XR or XDH protein. In yet other embodiments, the polynucleotide may comprise a dual or high order expression vector that encodes for an XR and an XDH polypeptide provided herein.

VI. Methods for Biological Nitrogen Fixation and Fertilization of a Plant

In one aspect, the present invention provides methods for the biological fixation of nitrogen. In certain embodiments, the methods comprise the use of an endophytic yeast capable of fixing atmospheric nitrogen. Endophytic yeast useful for nitrogen fixation include, for example, yeast isolated from within the stems of poplar (Populus) trees. In certain embodiments, these yeast strains are most closely related to Rhodotorula graminis or Rhodotorula mucilaginosa species. In a particular embodiment, the novel strains of the invention are selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1. In one embodiment of the invention, the novel endophytic yeast strains contain an rRNA gene sequence that is selected from any one of SEQ ID NOS:7 to 18. In a particular embodiment, an endophytic yeast strain of the invention may have an 18S rRNA gene sequence selected from SEQ ID NOS:7 to 9 or 16 to 18, an ITS rRNA gene sequence selected from SEQ ID NOS:10 to 12, or a 26S D1/D2 rRNA gene sequence selected from SEQ ID NOS:13 to 15.

In other embodiments, strain of yeast is a recombinant yeast harboring a heterologous gene sequence from an endophytic yeast strain selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1, wherein said strain is identified by an rRNA gene sequence selected from any one of SEQ ID NOS:7 to 18.

In certain embodiments, the heterologous gene sequence may be cloned into a microbial genome, for example a bacterial or yeast chromosome, or may comprise an expression vector, a recombinant or artificial microbial chromosome, for example a bacterial (BAC) or yeast (YAC) chromosome, a bacterial plasmid, a yeast plasmid, a recombinant bacteria phage, a recombinant viral vector, a mammalian expression vector, a baculovirus vector. In yet other embodiments, the heterologous gene sequence may encode for a fusion protein. In another embodiment, the heterologous gene sequence may encode for a tagged protein, such as a tagged XR or XDH protein.

In certain embodiments, the recombinant yeast strains may be a Saccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, a Brettanomyces, a Torulaspora, an Ascobotryozyma, a Citeromyces, a Debaryomyces, an Eremothecium, a Issatchenkia, a Kazachstania, a Kluyveromyces, a Kodamaea, a Kregervanrija, a Kuraishia, a Lachancea, a Lodderomyces, a Nakaseomyces, a Pachysolen, a Pichia, a Saturnispora, a Tetrapisispora, a Torulaspora, a Vanderwaltozyma, a Williopsis, and the like. In a particular embodiment, the recombinant yeast is a Saccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, or a Brettanomyces. In one embodiment, the strain is Saccharomyces cerevisiae. In a related aspect, biologically pure cultures of the recombinant yeast strains are provided.

In one embodiment, the invention provides a method for fertilizing a crop, the method comprising inoculating the crop with a strain of yeast capable of fixing nitrogen. In certain embodiments, the step of inoculating a crop comprises colonizing the soil the crop is planted in with the yeast. The soil may be colonized with the yeast prior to planting the crop, for example before, during, or after tilling the soil in preparation for planning the crop. In other embodiments, the soil may be colonized with the yeast after the crop has been planted.

The methods for fertilizing a crop with a nitrogen fixing yeast provided herein may, in certain instances, be used in conjunction or to supplement chemical fertilization or alternatively may replace chemical fertilization. For example, in certain embodiments the present invention provides a method for fertilizing a crop comprising inoculating the crop with a nitrogen fixing yeast in the absence of traditional fertilizer. In other embodiments, a method for fertilizing a crop is provided that comprises both inoculating the crop with a nitrogen fixing yeast and the use of a traditional chemical fertilizer. In certain embodiments, the amount of chemical fertilizer used may be less than would otherwise be used in the absence of a nitrogen fixing yeast, for example, at least about 5% less chemical fertilizer, or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 99% less chemical fertilizer than would otherwise be used in the absence of a nitrogen fixing yeast.

In some embodiments, the crop may be a food crop, including without limitation, sugar cane, maize, wheat, rice, potatoes, sugar beets, soybean, oil palm fruit, barley, tomato, coffee, cocoa, and the like. In certain embodiments, the crop may be a cereal grain, such as maize, rice, wheat barley, sorgum, millet, oats, rye, triticale, buckwheat, fonio, Quinoa, and the like; a vegetable, a melon, a root, a tuber, a fruit, a pulse, and the like.

In other embodiments, the crop may be a non-food crop, including without limitation, a crop grown for the production of a biofuel, such as a grass, a woody plant, a tree or shrub, such as a poplar, willow, or cottonwood, and the like; a crop used for building and or construction, such as hemp, wheat, linseed, flax, bamboo, and the like; a crop used for the production of a fiber, such as coir cotton, flax, hemp, manila hemp, papyrus, sisal, and the like; a crop used for the production of a pharmaceutical or recombinant protein, such as borage, Echinacea, Artemisia, tobacco, and the like; a crop used for the production of a biopolymer, such as wheat, maize, potatoes, and the like; a crop used for the production of a specialty chemical, such as lavender, oilseed rape, linseed, hemp, and the like.

DEFINITIONS

Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined.

As used herein, the term “endophytic yeasts” refers to fungi that reproduce asexually by budding from single cells, with absent or reduced hyphal states.

As used herein, “fermentation” refers to a process of breaking down and/or reassembling an organic substance. Fermentation may be either aerobic, anaerobic, or partially anaerobic (i.e. in the presence of low oxygen content). In the case of the present invention, fermentation generally refers to the production or conversion of an alcohol or a sugar alcohol, such as ethanol or xylitol, from a sugar or mixture of sugars, including pentose and hexose sugars.

As used herein, a “biologically pure culture” refers to a culture inoculated with a single microorganism or a sing strain of microorganism. Generally, the microorganism inoculated in a biologically pure culture may comprise at least about 50% of the total living mass of said culture. In certain embodiments, the microorganism may comprise at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the living mass of a biologically pure culture.

As used herein, a “lignocellulosic biomass” refers to biomass comprising cellulose, hemicellulose, and lignin. Many sources of lignocellulosic biomass are used for industrial fermentation, for example, wood residues (e.g., sawmill and paper mill discards), municipal wastes (e.g., newspaper and paper wastes), agricultural residues (e.g., corn stover, sugarcane bagasse, animal manures, cereal or flax straw, fruit, vegetable, and nut crop), dedicated energy crops (e.g., woody grasses, wood such as willow or poplar, corn, millets, clover), and the like.

The term “nucleic acid molecule” or “polynucleotide” refers to a deoxyribonucleotide or ribonucleotide polymer in either single-stranded or double-stranded form. It will be understood that when a nucleic acid molecule is represented by a DNA sequence, this also includes RNA molecules having the corresponding RNA sequence in which “U” (uridine) replaces “T” (thymidine).

The term “recombinant nucleic acid molecule” refers to a non-naturally occurring nucleic acid molecule containing two or more linked polynucleotide sequences. A recombinant nucleic acid molecule can be produced by recombination methods, particularly genetic engineering techniques, or can be produced by a chemical synthesis method. A recombinant nucleic acid molecule may include a protein of interest, such as a protein identified as useful in the production of xylitol or ethanol. The term “recombinant host cell” refers to a cell that contains a recombinant nucleic acid molecule. As such, a recombinant host cell can express a polypeptide from a “gene” that is not found within the native (non-recombinant) form of the cell.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

Reference to a polynucleotide “encoding” a polypeptide, protein, or enzyme means that, upon transcription of the polynucleotide and translation of the mRNA produced therefrom, a polypeptide is produced. The encoding polynucleotide is considered to include both the coding strand, whose nucleotide sequence is identical to an mRNA, as well as its complementary strand. It will be recognized that such an encoding polynucleotide is considered to include degenerate nucleotide sequences, which encode the same amino acid residues. Nucleotide sequences encoding a polypeptide can include polynucleotides containing introns as well as the encoding exons.

The term “expression control sequence” refers to a nucleotide sequence that regulates the transcription or translation of a polynucleotide or the localization of a polypeptide to which to which it is operatively linked. Expression control sequences are “operatively linked” when the expression control sequence controls or regulates the transcription and, as appropriate, translation of the nucleotide sequence (i.e., a transcription or translation regulatory element, respectively), or localization of an encoded polypeptide to a specific compartment of a cell. Thus, an expression control sequence can be a promoter, enhancer, transcription terminator, a start codon (ATG), a splicing signal for intron excision and maintenance of the correct reading frame, a STOP codon, a ribosome binding site, or a sequence that targets a polypeptide to a particular location, for example, a cell compartmentalization signal, which can target a polypeptide to the cytosol, nucleus, plasma membrane, endoplasmic reticulum, mitochondrial membrane or matrix, chloroplast membrane or lumen, medial trans-Golgi cisternae, or a lysosome or endosome. Cell compartmentalization domains are well known in the art and include, for example, a peptide containing amino acid residues 1 to 81 of human type II membrane-anchored protein galactosyltransferase, or amino acid residues 1 to 12 of the presequence of subunit IV of cytochrome c oxidase (see also, Hancock et al., EMBO J., 10:4033-4039 (1991); Buss et al., Mol. Cell. Biol., 8:3960-3963 (1988); U.S. Pat. No. 5,776,689, each of which is incorporated herein by reference).

The term “polypeptide” or “protein” refers to a polymer of two or more amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term “recombinant protein” refers to a protein that is produced by expression of a nucleotide sequence encoding the amino acid sequence of the protein from a recombinant DNA molecule.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, -carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (Ala, A), Serine (Ser, S), Threonine (Thr, T); 2) Aspartic acid (Asp, D), Glutamic acid (Glu, E); 3) Asparagine (Asn, N), Glutamine (Gln, Q); 4) Arginine (Arg, R), Lysine (Lys, K); 5) Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met, M), Valine (Val, V); and 6) Phenylalanine (Phe, F), Tyrosine (Tyr, Y), Tryptophan (Trp, V).

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/, or the like). Such sequences are then said to be “substantially identical” or “substantially similar.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is about 50, 100, 200, 300, 400, 500, or more amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. In certain embodiments, a comparison window may be at least about 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, or more positions. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math., 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol., 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA, 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds., Wiley Interscience (1987-2005)).

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

A subject nucleotide sequence is considered “substantially complementary” to a reference nucleotide sequence if the complement of the subject nucleotide sequence is substantially identical to the reference nucleotide sequence. The term “stringent conditions” refers to a temperature and ionic conditions used in a nucleic acid hybridization reaction. Stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5° C. to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature, under defined ionic strength and pH, at which 50% of the target sequence hybridizes to a perfectly matched probe.

The term “isolated” or “purified” refers to a strain, such as a yeast strain, or material, such as a protein or nucleic acid, that is substantially or essentially free from components that normally accompany the material in its native state in nature. Purity or homogeneity generally are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis, high performance liquid chromatography, rRNA gene sequencing, and the like. A yeast strain, polynucleotide, or polypeptide is considered to be isolated when it is the predominant species present in a preparation. Generally, an isolated protein or nucleic acid molecule represents greater than 80% of the macromolecular species present in a preparation, often represents greater than 90% of all macromolecular species present, usually represents greater than 95%, of the macromolecular species, and, in particular, is a polypeptide or polynucleotide that purified to essential homogeneity such that it is the only species detected when examined using conventional methods for determining purity of such a molecule. Generally, an isolated yeast strain represents greater than 50% of all microbiological species present in a sample, oftentimes an isolated yeast strain will represent greater than 75%, or greater than about 80%, 85%, 90%, 95%, of more of all microbiological species present in a sample.

EXAMPLES Example 1 Isolation of Endophytic Yeast from Poplar Stems

One yeast strain isolated from stems of wild cottonwood (Populus trichocarpa) in Three Forks Park at the Snoqualmie River near the towns of North Bend and Snoqualmie, King County, Wash. was named wild poplar strain 1 (WP1). Two yeast strains isolated from stems of hybrid poplar (Populus trichocarpa×P. deltoides) in greenhouses at the University of Washington, Seattle, and Oregon State University, Corvallis were named as PTD2 and PTD3, respectively. The poplar stems were surface-sterilized with 10% bleach (1.2% active sodium hypochloride) for 10 minutes and 1% iodophor for 5 minutes, and then rinsed for 3-5 times with sterile water. The ends of the explants were removed, and stems were incubated in the light on Murashige and Skoog medium (MS; Caisson 61 laboratories Inc., Rexburg, Id.). Morphologically-distinct colonies were streak purified on YPD (Yeast extract, Peptone, and Dextrose) plates.

Rhodotorula glutinis strain ATCC 2527, obtained from American Type Culture Collection (ATCC), and a Baker's yeast Saccharomyces cerevisiae Meyen ex E.C. Hansen strain (Lesaffre yeast corporation, Milwaukee, Wis.) were used for comparison. The phylogenetic relatedness in the three rRNA genes: 18S, 26S (D1/D2 domains), and ITS, as well as their phenotypic characteristics was examined. The capacity to produce IAA by the three yeast strains was also examined.

FIG. 1 shows photomicrographs of the three yeast strains and R. glutinis ATCC in YPD broth. WP1 cells (1A) were ovoid to subspherical, subhyaline, vacuolate, budding on one end, and 6.5-9×5-8 μm. PTD2 cells (1B) were broadly ovoid, subhyaline, vacuolate, budding on one end, and 5-7×3-4 μm. Strain PTD3 cells (1C) were ovoid to subspherical, subhyaline, vacuolate, exhibited budding on one end, and 4-5.5×3-4 μm. R. glutinis (ATCC strain) cells (1D) were ellipsoid to ovoid, sub-olivaceous to sub-hyaline, budding at one or both ends, forming pseudohyphae, and 4.5-7.5×3-5 μm. All strains formed colonies of varying shades of pink on YPD agar plates at 30° C. Sexual reproduction was not observed in any of the three Populus isolates during culturing for one-week period at 30° C.

Example 2 Extraction of Yeast Genomic DNA

Genomic DNA of yeast was prepared according to the rapid isolation of yeast chromosomal DNA protocol (Ausubel et al., 1995) with modifications. Yeast cultures grown overnight in 10 mL YPD broth at 30° C. were collected by centrifuging at 3000×g for 5 min under room temperature and washed with 1 mL sterile DI H₂O. Cells were lysed by vortexing with 0.5 g glass beads in 1 mL breaking buffer and 1 mL phenol/chloroform/isoamyl alcohol at high speed for 3 min. The water layer was separated by centrifugation, transferred, and washed through multiple phenol/chloroform extraction steps. Extracted DNA was then precipitated using an equal amount of isopropanol at room temperature. Resuspended DNA in TE buffer was stored at −20° C.

Example 3 PCR Amplification of 18S, ITS, and D1/D2 Region

The present example focuses on the phylogenetic relatedness of three rRNA genes: 18S, 26S (D1/D2 domains), and ITS, from the novel isolated yeast strains

Yeast DNA was purified and amplified with PCR using three sets of primers for 18S, ITS, and D1/D2 region of rRNA genes, respectively. The primers used in this study are listed in Table 1. A 1.8-kb fragment of 18S rRNA gene was amplified with primers NS8 and NS1. A 600-650 bp fragment of D1/D2 region at the 5′ end of the large-subunit rRNA 84 gene was amplified with primers F63 and LR3. A 600-620 bp fragment of ITS1-5.8S-ITS2 region on the rRNA gene was amplified with primers ITS1 and ITS4. PCR was performed on DNA extracts in 25 μl with final concentrations of 1×PCR Pre-Mix buffer E (Epicentre, Madison, Wis.), 100 nM of forward and reverse primers, 5 U of Taq DNA polymerase (Fermentas), and 1 μL of template DNA. The reaction mixture was held at 95° C. for 5 minutes followed by 34 cycles of amplification at 95° C. for 30 s, annealing temperature as shown in Table 1 for 30 s and 72° C. for 60 s, with a final step of 72° C. for 5 minutes in a Mastercycler thermalcycler (Eppendorf, Westbury, N.Y.).

TABLE 1 Primers used for the PCR amplification of 18S, ITS, and  D1/D2 genomic rRNA regions. Annealing SEQ temperature, Primer Sequence (5′-3′) ID NO: ° C. Reference NS8 FP TCC GCA GGT TCA CCT ACG GA 1 44 White et al., 1990 NS1 RP GTA GTC ATA TGC TTG TCT C 2 44 White et al., 1990 F63 FP GCA TAT CAA TAA GCG GAG GAA AAG 3 45 Fell et al., 2000 LR3 RP GGT CCG TGT TTC AAG ACG G 4 45 Fell et al., 2000 ITS1 FP TCC GTA GGT GAA CCT GCG G 5 44 White et al., 1990 ITS4 RP TCC TCC GCT TAT TGA TATG C 6 44 White et al., 1990

Example 4 Molecular Cloning and Sequencing

PCR products were subjected to electrophoresis in 0.8% agarose gel. Target bands were collected from the agarose gel and DNA extracted from it using the QIAEXII gel extraction kit (Qiagen, Madison, Wis.). DNA fragments were cloned using the pGEM T Easy kit (Promega, Madison, Wis.) following the manufacturer's instructions. Sequencing was conducted using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and an ABI 3730XL sequencer (Applied 100 Biosystems) at the Department of Biochemistry sequencing facility of the University of Washington. Sequence data have been submitted to Genbank under the accession numbers EU563924-EU563932.

TABLE 2 Genomic rRNA sequences. Accession SEQ Strain Gene number ID NO: WP1 18S ribosomalRNA EU563924 7 PTD2 18S ribosomalRNA EU563925 8 PTD3 18S ribosomalRNA EU563926 9 WP1 ITS ribosomal RNA EU563927 10 PTD2 ITS ribosomal RNA EU563928 11 PTD3 ITS ribosomal RNA EU563929 12 WP1 26S D1/D2 ribosomal RNA EU563930 13 PTD2 26S D1/D2 ribosomal RNA EU563931 14 PTD3 26S D1/D2 ribosomal RNA EU563932 15

Example 5 Analysis of DNA Sequences

DNA sequences were aligned with the program ClusterW (Thompson et al., 1994) using default gap penalties. The selection of sequences for construction of phylogenetic trees was done by comparing the target sequences to all sequences in the GenBank by the online BLAST program. Phylogenetic trees were constructed using the neighbor-joining distance method (Saitou and Nei, 1987) and distances computed using the Jukes-Cantor methods (Jukes and Cantor, 1969) and using the Maximum Parsimony method (Eck and 111 Dayhoff, 1966). All analyses were conducted with the program MEGA 4 (Tamura et al., 2007).

Analysis of the 18S, ITS1-5.8S-ITS2, and D1/D2 regions suggested that isolates PTD2 and PTD3 were most closely related to Rhodotorula mucilaginosa (FIGS. 2-7). PTD2 was identical to R. mucilaginosa in both the 18S and D1/D2 region sequences, but differed from R. mucilaginosa in 5 of 183 base positions when the ITS1-5.8S-ITS2 sequences were compared. PTD3 was identical to R. mucilaginosa in the D1/D2 region, and differed from R. mucilaginosa in 1 of 586 bases in the 18S sequence and 7 of 583 bases in the ITS1-5.8S-ITS2 sequence.

Sequences from WP1 appeared to support relationships with several different species. Rhodosporidium babjevae and WP1 shared the most similar 18S rRNA gene sequences (FIG. 2), while the ITS1-5.8S-ITS2 and D1/D2 sequences of WP1 were most similar to those of Rhodotorula graminis and Rhodotorula glutinis (FIGS. 3 and 4), respectively. The ITS1-5.8S-ITS2 sequence of WP1 differed from R. glutinis at 4 of 583 base positions (all in the ITS1 region), from R. graminis in 1 of 583 base positions (in the ITS1 region), and from that of R. babjevae in 6 of 583 positions (4 in ITS1, 1 in 5.8S, and 1 in ITS2). In the D1/D2 region, WP1 was identical to R. glutinis based on 586 positions, and differed from R. graminis in 1 and from R. babjevae in 2 base positions. In the 18S, WP1 was identical to R. babjevae based on 952 positions, and differed from R. glutinis by 1/952. It should be noted that the WP1 18S sequence was identical to the sequence of a R. graminis strain in GenBank (Accession number X83827) but the R. graminis sequence contained missing data for 7 positions. The strain was not included in the phylogenetic tree shown in FIGS. 2 and 5.

Example 6 IAA Production Test

To quantify the production of IAA, isolates were grown in YPD/YMA medium with or without 0.1% (w/v) L-tryptophan for 1, 2, 5, and 7 days and 1.5 mL of the cells were pelleted by centrifugation at 10,000×g for 5 min. One mL of supernatant was mixed with 2 mL of Salkowski reagent (2 mL of 0.5 M FeCl₃+98 mL 35% HClO₄) (Gordon and Weber, 1951), and the intensity of pink color developing in the mixture after 30 min was quantified by a Hach DR/4000 spectrophotometer (Hach, Loveland, Colo.) at wavelength 530 nm. Cell pellets were dried at 100° C. overnight and weighed for normalizing IAA production. Similarly, pink color was also developed for a series of IAA standard solutions to establish a stand curve.

No detectable IAA was produced for all tested yeast strains after 7-day incubation without L-tryptophan. When incubated with 0.1% L-tryptophan, strains of WP1, PTD2, PTD3, and R. glutinis ATCC showed significant production of IAA (FIG. 9). No detectable IAA was produced by Baker's yeast. The overall production of IAA increased with time for the four yeast strains. Among them, WP1 had the highest IAA production and PTD3 had the least

Example 7 Phenotypic Characterization of Yeast Strains

The morphology of yeast strains was determined and photographs made using a Leica DMR compound microscope equipped with brightfield and differential interference contrast optics and a Leica DC300 digital camera (Leica Microsystems GmbH, Wetzlar). Utilization of kinds of carbon sources was examined using a commercial API 20C AUX yeast identification kit (bioMerieux, Durham, N.C.) according to the manufacture's instructions. Yeast cultures in YPD broth diluted to an optical density (OD₆₀₀) of 0.451, which is equivalent to McFarland standard No. 2, in 0.85% NaCl solution were applied to the API 20C AUX strips. The strips then were incubated at 30° C. Pink color developed on the incubation strips at 48 and 72 hours to indicate utilization of individual carbon sources by tested yeast strains.

A commonly used method to distinguish many yeast species is comparison of their abilities to utilize certain organic compounds as the sole major source of carbon (Barnett et al., 2000). A commercial yeast identification kit, API 20C AUX, can identify rapidly common and rare clinical yeast isolates with high efficacy (Ramani et al., 1998; Verweij et al., 1999). Table 3 summarizes utilization of 19 different organic compounds by the three Populus isolates and two controls (R. glutinis ATCC and Baker's yeast), with the API 20C AUX system. Characteristics of R. graminis were compiled from the literature (Barnett et al. 2000).

TABLE 3 Summary of utilization of 19 carbon sources by strains WP1, PTD2, PTD3, ATCC, and Baker's yeast assessed using the API 20C AUX system. R. glutinis Baker's Carbon Source WP1 PTD2 PTD3 ATCC yeast R. graminis D-glucose + + + + + + Glycerol + + − − − + Calcium + − − + − V 2-keto- gluconate L-arabinose − + + − − +, D D-xylose + + + − − + Adonitol + + + − − NA Xylitol − + + − − +, D D-galactose + − + + + + Inositol − − − − − − D-sorbitol + + + + − NA Methyl- − − − − + NA αD-gluco- pyranoside N-acetyl- − − − − − − glucosamine D-cellobiose − − − − − + D-lactose − − − − − − D-maltose − + + + + V Sucrose + + + + + + D-trehalose − + + − + +, D D-melezitose − V − + + − D-raffinose + + + + − + The profile for R. graminis was compiled from Barnett et al. (2000). “+”—positive, “−”—negative, “V”—variable, “NA”—not available, “D”—delayed longer than 7 days

The clusterings between Baker's yeast and S. cerevisiae and between R. glutinis ATCC and R. glutinis in FIG. 8 demonstrated that the identification system was effective. Similarity of PTD2, PTD3, and R. mucilaginosa (FIG. 8) carbon utilization profiles confirmed the groupings based on rRNA gene sequences. However, WP1 did not cluster with any Rhodotorula species in the reference list of the API 20C AUX system (FIG. 8) and diverged in utilization of 5 compounds (glycerol, D-xylose, adonitol, D-maltose, and D-melezitose) when compared with R. glutinis ATCC. Compared to the reported carbon-utilization profile from the literature, WP1 diverged from R. graminis only in utilizing D-cellobiose out of 13 organic compounds. Regarding another 3 compounds (L-arabinose, xylitol, and D-trehalose) labeled positive for R. graminis in Table 2 with over 7-day delayed observation, direct comparison to the WP1 profile obtained after 3-day incubation with API 20C AUX system is not proper.

Example 8 Clustering Analysis of Phenotypic Characteristics of Yeast Strains

A clustering map was drawn by JMP statistics software version 6 (SAS, Cary, N.C.) according to the Ward's minimum variance method (Milligan, 1980). Distance for Ward's method is determined according to the formula:

$D_{KL} = \frac{{{{\overset{\_}{x}}_{K} - {\overset{\_}{x}}_{L}}}^{2}}{\frac{1}{N_{K}} + \frac{1}{N_{L}}}$

wherein X_(K) and X_(L) are the mean vectors for cluster C_(K) and C_(L), respectively, C_(K) is the K^(th) cluster and C_(L) is the L^(th) cluster; N_(K) and N_(L) are the numbers of observations in C_(K) and C_(L).

Three pink-pigmented yeast strains isolated from stems of Populus grew well on YPD medium under aerobic conditions. Phylogenetic analysis of rRNA gene sequences supported determination of the yeast strains, PTD2 and PTD3 as Rhodotorula mucilaginosa. Determination of WP1 was not as simple as that of PTD2 and PTD3 since analyzing different sequence data provided differing results. Rhodotorula and Rhodosporidium are members of the class Urediniomycetes of phylum Basidiomycota. Rhodotorula glutinis, R. graminis, R. babjevae, and R. mucilaginosa grouped together in the Sporidiobolus clade, based on phylogenetic analysis of ITS and D1/D1 regions (Fell et al., 2000; Scorzetti et al., 2002). Rhodotorula glutinis, R. graminis, and R. babjevae occurred on the same branch of the Sporidiobolus clade, suggesting a close phylogenetic relationship among them. As the ITS region is generally considered to be less conserved than either small or large subunits of rRNA genes (Scorzetti et al., 2002), the ITS analysis could be more informative in distinguishing close related species. A single substitution out of 583 positions in the ITS1-5.8S-ITS2 between WP1 and R. graminis compared to 4 substitutions for WP1 and R. glutinis and to 6 substitutions for WP1 and R. babjevae suggests that WP1 is more closely related to R. graminis. In addition, WP1 shared higher similarity on carbon-utilization profiles with R. graminis than R. glutinis. Based on the phylogenetic and phenotypic characteristics of WP1, we regard the WP1 isolate as most closely fitting the current concept of R. graminis.

Rhodotorula mucilaginosa has been isolated from a wide variety of sources, including the bark of Quercus suber L. (cork oak) (VIIIa-Carvajal et al., 2004), soil and mosses from Antarctica (Pavlova et al., 2001), food stuffs (Haridy, 1993; Botes et al., 2007), and humans (Neofytos et al., 2007). The species has been reported frequently from wastewater treatment plants and exhibited tolerance to heavy metals such as copper, cadmium, and uranium (de Siloniz et al., 2002; Balsalobre et al., 2003; Villegas et al., 2005). Epoxide hydrolase of R. mucilaginosa can hydrolyze glycidyl ethers (Kotik et al., 240 2005), dibenzofuran (Romero et al., 2002), and other benzene compounds (Middelhoven 241 et al., 1992) in environmental bioremediation processes. Rhodotorula graminis was first isolated from the leaf surfaces of pasture grasses (di Menna, 1958) and later found widely in the environment, being isolated from soil (Vadkertiova and Slavikova, 1994; Hobbie et 244 al., 2003), Ceratonia siliqua L. (carob trees) (Spencer et al., 1995), and tropical fruits (Trindade et al., 2002). The species has shown an ability to cleave aromatic rings (Durham et al., 1984) and has been studied for the bioremediation of benzene compounds (Middelhoven, 1993).

Recently, the role of endophytes in phytoremediation of xenobiotics has been highlighted, including increasing plant tolerance to heavy metals (Lodewyckx et al., 2001), reducing phytotoxicity of herbicides (Germaine et al., 2006), and facilitating degradation of nitro-aromatic compounds (van Aken et al., 2004b). The tolerance to heavy metals and degradation of xenobiotics by R. mucilaginosa and R. graminis suggests the new Populus endophytic yeast strains may be suitable for phytoremediation applications. Furthermore, the production of IAA by the three yeast strains could potentially promote plant growth.

The three yeast strains produced IAA only with the addition of L-tryptophan. As one of the most expensive standard protein amino acids, in terms of energy, to produce (Hrazdina and Jensen, 1992), tryptophan is not biosynthesized by all bacteria and yeasts. Those microorganisms incapable of synthesizing tryptophan have to rely on their plant hosts or surrounding microbial sources (Radwanski and Last, 1995). With tryptophan available in the Populus tissue, the endophytic yeasts do not have to spend high energy on synthesis of the amino acid by themselves. At the same time, the ability to convert tryptophan to IAA by the endophytes would, in return, benefit the tryptophan provider, which may be seen as a mutually advantageous plant-microbe example.

To our knowledge, the yeast strains provided by the present invention are the first endophytic yeast strains isolated from species of Populus. The strain from wild Populus, WP1, has been chosen for whole genome sequencing by the Joint Genome Institute of the Department of Energy due to its potential applications for bioenergy production. The determination and characterization presented in the present invention should benefit future research on these strains.

Example 8 Growth Requirement Test

In order to study the sugar utilization of the endophytic yeast strains, WP1, PTD3 and the baker's yeast (BK), isolates were streaked from frozen glycerol stocks onto yeast extract, peptone, dextrose (YPD) agar to obtain isolated colonies. A single colony was transferred to 10 ml of YPD broth and incubated on a shaker at 30° C. overnight. The overnight culture was harvested and washed with MS medium (Caisson Labs MSP009) twice. For growth curve assays, cells were grown in 25 ml of MS medium containing either 3% glucose or 3% xylose at pH 5.8. Growth was monitored using a spectrophotometer measuring the optical density at 600 nm (OD600). Statistical analysis was done using split plot ANOVA (Intercooled Stata 10.0, StataCorp LP, College Station, Tex.) in order to account for the multiple measures taken over time on each flask, and the replicated flasks for each sample.

In order to study the sugar utilization of the two endophytic yeast strains, WP1 and PTD3, growth rate was monitored in media with different sugars. The growth curve experiments showed that both WP1 and PTD3 grew well in glucose (FIG. 34A) and xylose (FIG. 34B) sugars. As reported previously, Baker's yeast did not utilize xylose. It is also noteworthy that PTD3 grew better than WP1 under the two conditions and PTD3 was a better xylose utilizer (FIG. 34B). There was about a 24 hour-delay before WP1 and PTD3 started growing in glucose and xylose. The delay was most likely from the shift from rich medium (YPD) to minimal medium (plain MS).

Example 9 Cloning of the Xylose Reductase (XR) and Xylitol Dehydrogenase (XDH) Encoding Genes XYL1 and XYL2 from WP1

Yeast strain WP1 was isolated from stems of wild cottonwood (Populus trichocarpa) and was identified as Rhodotorula graminis (Xin et al. 2009). Another yeast strain, PTD3, was isolated from stems of hybrid poplar (Populus trichocarpa×P. deltoides) and was identified to be species Rhodotorula mucilaginosa (Xin et al. 2009). A baker's yeast ATCC6037 strain was used as the control yeast.

WP1 and PTD3 genomic DNA was prepared following a published protocol (Burke et al. 2000) with the following modifications: two extra phenol:chloroform/chloroform extractions and isopropanol precipitation were carried out. For mRNA preparation, cells were grown in YPD, which was prepared as described (Kaiser et al. 1994) except that sugars were autoclaved separately from the basal medium. YPX and YPGX were similar to YPD but replaced dextrose with xylose or xylose plus glucose. Isolation of mRNA was performed by the method described in (Laplaza et al. 2006).

Isolated RNA was quantified using a NanoDrop spectrophotometer (ND1000). Reverse transcription (RT) and subsequent PCR amplifications were performed sequentially using the OneStep RT-PCR Kit (QIAGEN). The whole WP1 XR and XDH-encoding genes were amplified by RT-PCR using two sets of primers (WP1-XR-F, WP1-XR-R and WP1-XDH-F, WP1-XDH-R), which were designed based on the sequences of XYL1 and XYL2 genes in Pichia stipitis (GenBank accession numbers: CAA42072, AAD28251) as well as the alignment results with WP1 whole genome sequence (sequencing by JGI and is available online) with the following modifications.

The genome sequence of WP1 was provided through the DOE Joint Genome Institute sequencing effort (see Acknowledgements). Putative XYL1 and XYL2 genes were first found in the JGI sequence using BLAST and the resulting sequences were utilized to design primers for the cloning of the mRNA sequences of the two genes from WP1. Sequence comparisons of the cloned genes with public databases were performed via the Internet at the National Center for Biotechnology Information site (http://www.ncbi.nlm.nih.gov/), by employing the tblast algorithm (Altschul et al. 1997). GenomeScan (Chris Burge, Biology Dept. at MIT http://genes.mit.edu/genomescan.html) was employed to predict the gene exon/intron structures and putative XR and XDH mRNA sequences in WP1. All the resulting sequences in WP1 and PTD3 were aligned with homologous protein sequences of other D-xylose-fermenting yeasts (e.g. Pichia stipitis, Candida. spp) using the local BLAST program (B12seq).

The resulting PCR products were purified using the QIAEXII gel extraction kit (Qiagen, Madison, Wis.) and then inserted into the pGEM-T Easy vector (Promega, Madison, Wis.) following the manufacturer's instructions. Sequencing of the inserts in both directions was performed by the UW Biochemistry Department Sequencing Facility using the BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems).

TABLE 4 Primers used for cloning of the XR and XDH-encoding genes from WP1 and PTD3 and in expression studies thereof. SEQ ID Primer Sequence NO: WP1-XR-F ATGGTCCAGACTGTCCCC 19 WP1-XR-R TCAGTGACGGTCGATAGAGATC 20 WP1-XDH-F ATGAGCGCTCCCAGTCTCGC 21 WP1-XDH-R TCACTCGAGCTTCTCGTCGAC 22 PTD3-D-XR-F GCYATCAAGKCGGGYTACCG 23 PTD3-D-XR-R GTGGWAGBTGTTCCASAGCTT 24 PTD3-D-XDH-F CCMATGGTCYTSGGNCACGA 25 PTD3-D-XD-R CCGACVGGVCCDGCDCCAAAGAC 26 PTD3-XR-GSP1 GCCAGTGGATGAGGTAGAGG 27 (for 5′ RACE) PTD3-XR-GSP2(for GTGATGAAGATGTCCTTGCG 28 5′ RACE) PTD3-XR-GSP3(for AGGTCTACGGCAACCAGAAG 29 3′ RACE) PTD3-XR-GSP4(for ATCACCTCGAAGCTCTGGAAC 30 3′ RACE) PTD3-XDH-GSP1 GATGAGCGATTTGAGGTTGAC 31 (for 5′ RACE) PTD3-XDH-GSP2 CCTTGGCAACTGCGTGGAC 32 (for 5′ RACE) PTD3-XDH-GSP3(for GCAAAGGTGGTCATTACGAAC 33 3′ RACE) PTD3-XDH-GSP4(for CTCCTTGAGCCCATGTCGGT 34 3′ RACE) #XR-F ATCACCTCGAAGCTCTGGAAC 35 #XR-R GCCAGTGGATGAGGTAGAGG 36 #XDH-F CTCCTTGAGCCCATGTCGGT 37 #XDH-R GATGAGCGATTTGAGGTTGAC 38 515F(18S rRNA) GTGCCAAGGCAGCCGCGGTAA 39 1209R(18S rRNA) GGGCATCACAGACCTG 40 note: K = G/T V = A/C/G M = A/C N = A/C/G/T R = A/G B = C/G/T S = C/G W = A/T Y = C/T D = A/T/G

The XR and XDH-encoding genes were cloned and sequenced from WP1 using primers based on the genomic sequence of WP1 provided by the DOE JGI sequencing project. Analysis of the two genes was then performed on the cloned sequences (not directly from the JGI sequences provided). The 1259 nucleotide sequence of WP1-XR contains an open reading frame of 987 nucleotides (SEQ ID NO:41) encoding a polypeptide of 322 amino acids (SEQ ID NO:42). The 1216 nucleotide sequence of WP1-XDH contains an open reading frame of 1191 nucleotides (SEQ ID NO:43) encoding a polypeptide of 396 amino acids (SEQ ID NO:44). At the amino acid level, the WP1-XR gene is 37% and 36% identical to XYL1 gene of Pichia stipitis (XP_(—)001385181) and Candida guilliermondii (O94735), respectively; the WP1-XDH gene is slightly more conserved: 41% identity to XYL2 gene of Pichia stipitis (XP_(—)001386982) and Candida tropicalis. The visualized annotation pictures (by using vector NTI10) of the two genes show that both XR and XDH genes are more complex than those of Pichia stipitis which has no introns in the genes (FIGS. 35 and 36) (Amore et al. 1991).

Example 10 WP1 XR and XDH Gene Expression Levels in Glucose and Xylose

To investigate the XR and XDH gene expression in WP1, cells were grown in medium containing either glucose or xylose, and the RNA was purified from the cultures. Segments from the mRNA were amplified using RT-PCR with primers specific for each of the two genes. As shown in FIG. 37, the XR and XDH genes were expressed in WP1 cells grown in xylose. These results indicated that the genes are indeed transcribed and that XR and XDH gene expression was upregulated by xylose. The XR gene expression was not detectable when the cells were grown in glucose; however, there was some low-level constitutive expression of the XDH in glucose.

Briefly, total RNA was isolated from cells grown in media containing 2% glucose, 2% xylose, 1% glucose+1% xylose or 2% glucose+2% xylose respectively. RT-PCR was applied on the same amount of total RNAs from different media using the primer sets #XR-F, #XR-R and #XDH-F, #XDH-R (Table 4) designed to work equally well for both WP1 and PTD3. To verify that the same amount of total RNA was used, 18S rRNA semiquantitative RT-PCR was performed in WP1 under these different culture conditions using primer set 515F and 1209R (downloaded from JGI for eukaryotic 18S rRNA gene amplification).

Example 11 Cloning of the Xylose Reductase (XR) and Xylitol Dehydrogenase (XDH) Encoding Genes XYL1 and XYL2 from PTD3

For cloning the partial XR and XDH-encoding genes in PTD3 (genome sequences are not available), RT-PCR was performed using the degenerate primers PTD3-D-XR-F, PTD3-D-XR-R and PTD3-D-XDH-F PTD3-D-XDH-R, which were designed based on the multiple sequence alignment amongst PTD3, WP1 and other D-xylose-fermenting yeasts (CLUSTALW, Thompson et al. 1994). Following RT-PCR, samples were subjected to electrophoresis in a 1% agarose get, using Sybersafe (Invitrogen) as a DNA intercalating and visualizing agent, at 100V for 1 hour.

Since strain PTD3 was a more effective utilizer of xylose compared to WP1, the xylose metabolism genes where cloned from this strain. However, the PTD3 genome has not been sequenced, so a different approach was used to clone the two genes than was used for WP1. The partial PTD3 XR and XDH-encoding genes were cloned using degenerate primer sets that were designed based on the multiple sequence alignment amongst PTD3, WP1 and other D-xylose-fermenting yeasts (Table 4). The complete nucleotide sequences were subsequently determined by 5′ and 3′ rapid amplification of cDNA ends (RACE) using gene specific primers based on the cDNA fragment sequences. Briefly, the partial PTD3 XR and XDH-encoding genes were amplified by RT-PCR and sequenced, and the complete nucleotide sequences were subsequently determined by 5′ and 3′ rapid amplification of cDNA ends (RACE) using a 5′/3′ RACE kit (FirstChoice RLM-RACE Kit, Applied Biosystems). For 5′RACE, the gene-specific primers PTD3-XR-GSP1, PTD3-XR-GSP2, PTD3-XDH-GSP1 and PTD3-XDH-GSP2 were used. For 3′ RACE, the gene-specific primer PTD3-XR-GSP3, PTD3-XR-GSP4, PTD3-XDH-GSP3 and PTD3-XDH-GSP4 were used. Primer sequences are listed in Table 4.

The 1087 bp nucleotide sequence of the cloned PTD3-XR contained an open reading frame of 975 bp nucleotides (SEQ ID NO:45) encoding a polypeptide of 324 amino acids (SEQ ID NO:46). The alignment results show that PTD3-XR protein is 67% identical to the WP1 XR protein (Table 5). The 1409 bp nucleotide sequence of PTD3-XDH contains an open reading frame of 1185 bp nucleotides (SEQ ID NO:47) encoding a polypeptide of 394 amino acids (SEQ ID NO:48). The alignment results showed that PTD3-XDH protein is 69% identical to the WP1 XDH protein. Alignments with other yeasts were also performed to study the homology with the two genes in PTD3 (Table 5). The XR and XDH proteins of WP1 and PTD3 were 69-73% identical, whereas they are only 37-41% identical to these proteins from other known xylose-utilizing species.

TABLE 5 XR and XDH identities between homologous proteins in several yeast strains. Pichia Candida Candida Identity WP1 stipitis guilliermondii tropilis PTD3 XR 73% 38% 37% 39% PTD3 XDH 69% 37% Null 41% GenBank Accession No.: XR: CAA42072 (P. stipitis); ABX60132 (C. tropicalis); AAD09330 (C. guilliermondii). XDH: AAD28251 (P. stipitis); ABB01368 (C. tropicalis). The XDH protein sequence of Candida guilliermondii was unavailable.

Example 12 PTD3 XR and XDH Gene Expression Levels in Glucose and Xylose

To investigate the expression of the two genes in PTD3, cells were grown in glucose and xylose media as with the WP1 study. PTD3 gene specific primers were used to amplify the segments from mRNA by using RT-PCR. Different bands corresponding to XR and XDH were observed in mRNA from cells grown on either glucose or xylose (FIG. 38). These results indicate that the genes are indeed transcribed within mRNA and that the XR and XDH gene expression was induced by xylose. As in WP1, the genes were barely expressed in medium containing only glucose as the carbon source.

Example 13 Comparison of the Gene Expression Levels of XR and XDH Between WP1 and PTD3

In order to better understand the differences in utilization of xylose between the two endophytic yeast strains, the expression levels of the XR and XDH genes were compared between the strains. Using the aligned WP1 and PTD3 sequences, primers were designed to the gene regions of identity so that the expression of XR and XDH-encoding genes could be directly comparable. RT-PCR was performed to amplify the mRNA segments from WP1 and PTD3 cells grown in YP medium containing different sugars (2% glucose, 2% xylose, 1% glucose+1% xylose, 2% glucose+2% xylose). As shown in FIG. 39, both the XDH and XR genes are expressed to higher levels in PTD3 than in WP1 when the yeast were grown in xylose medium. The expression of the two genes appeared slightly suppressed in 1% xylose+1% glucose medium compared to 2% xylose medium.

In order to investigate whether the expression differences resulted from the lower xylose concentration in the mixed sugar medium or from repression by glucose, an RT-PCR experiment was also conducted under 2% glucose+2% xylose culture condition. As shown in (FIG. 40) in WP1, the expression of the two genes were still slightly suppressed in 2% xylose+2% glucose medium compared to 2% xylose medium. However, the gene expression was not suppressed by glucose in PTD3. In this strain, the level of the XR and XDH gene expression was about equal in both the mixed sugar medium and the xylose medium. 18S rRNA RT-PCR was performed as an internal control showing that equal amounts of total RNA were used under these different culture conditions (FIG. 41).

Example 14 Nitrogen Fixation of Endophytic Yeast

To determine if any of the isolated endophytic yeast strains could fix atmospheric nitrogen, several isolated strains were incubated in nitrogen limiting media (NFM). Surprisingly, it was found that WP1, as well as two other pink yeasts isolated from greenhouse-grown poplar hybrids, were among the endophytes that grew well on NFM. Amplification of the nifH gene using universal primers indicated that these isolates contain the nitrogenase gene required for nitrogen fixation. FIG. 33 shows the growth of WP1 and Saccharomyces cerevisiae (baker's yeast) in NFM as quantified by OD600. These results suggest that the isolated endophytic yeast strains provided herein are able to fix atmospheric nitrogen.

Example 15 Use of Endophytic Yeast for Nitrogen Fixation and Supplementation of Nitrogen Deficiencies for Plant Growth

To determine if nitrogen fixing yeast could be used to promote plant growth under nitrogen limiting conditions, corn was grown for 11 weeks in soil without nitrogen supplementation in the presence (WP1) or absence (non-symbiotic; NS) or the nitrogen fixing strain WP1. As seen in FIG. 42, corn grown in the presence of the WP1 yeast strain consistently grew much more robustly, providing about 5 times more biomass (B(g)) than corn grown in the absence of WP1 (compare FIG. 42B with FIG. 42A, respectively). In addition, the % viability in WP1 colonized plants (58-92%) was higher than uninoculated plants (8.3-29.2%) plants. Statistical analysis indicated significant differences (P≦0.1) for both viability and biomass with WP1 symbiotic plants having higher viability and biomass compared to uninoculated plants. A graphic representation of these data are provided in FIG. 44. Thus, nitrogen fixing endophytic yeast strains isolated from within poplar trees can significantly promote the growth of corn, even in the absence of traditional nitrogen sources. As such, these yeast can be used for biological nitrogen fixation instead of chemical fertilizers to lower costs and reduce nitrous oxide emissions into the atmosphere.

REFERENCES

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It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. 

1. A method of producing ethanol, xylitol, or a mixture thereof, the method comprising; fermenting a carbon source with an endophytic strain of yeast selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1 wherein said strain is identified by an rRNA gene sequence selected from any one of SEQ ID NOS:7 to
 18. 2. The method of claim 1, wherein the carbon source is a five carbon sugar or sugar alcohol, or a six carbon sugar or sugar alcohol.
 3. (canceled)
 4. The method of any one of claim 1, wherein the carbon source is selected from the group consisting of glucose, glycerol, calcium 2-keto-gluconate, arabinose, xylose, adonitol, xylitol, galactose, inositol, sorbitol, methyl-α-glucopyranoside, N-acetyl-glucosamine, cellobiose, lactose, maltose, sucrose, trehalose, melezitose, raffinose, and combinations thereof. 5-6. (canceled)
 7. The method of claim 1, wherein said carbon source comprises lignocellulosic biomass, xylose, glucose, or a combination thereof.
 8. The method of claim 1, wherein said method comprises the steps of: (a) treating a source of lignocellulosic biomass to produce a mixture of pentose and hexose sugars; (b) separating a first stream comprising xylose from said mixture; (c) fermenting said first stream with the endophytic strain of yeast.
 9. The method of claim 8, further comprising fermenting a second stream comprising glucose with the endophytic strain of yeast.
 10. (canceled)
 11. The method of claim 1, wherein said method further comprises purifying ethanol, xylitol, or both after fermentation.
 12. The method of claim 11, wherein purification comprises first distilling ethanol and then purifying xylitol from the distillation residuals. 13-14. (canceled)
 15. A recombinant yeast strain capable of fermenting both a five carbon sugar and a six carbon sugar, the yeast harboring a heterologous gene sequence from an endophytic yeast strain selected from the group consisting of Rhodotorula graminis strain WP1, Rhodotorula mucilaginosa strain PTD2, Rhodotorula mucilaginosa strain PTD3, and Rhodotorula mucilaginosa strain Ad1, wherein said strain is identified by an rRNA gene sequence selected from any one of SEQ ID NOS:7 to
 18. 16. The recombinant yeast strain of claim 15, wherein the heterologous gene sequence encodes for a polypeptide comprising an amino acid sequence that is at least 85% identical to an amino acid sequence selected from SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.
 17. The recombinant yeast strain of claim 15, wherein the heterologous gene sequence encodes for a polypeptide comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.
 18. The recombinant yeast strain of claim 15, wherein the heterologous gene sequence encodes for a polypeptide comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, and SEQ ID NO:48.
 19. The recombinant yeast strain of claim 15, wherein the yeast is a Saccharomyces, a Schizosaccharomyces, a Candida, a Zygosaccharomyces, or a Brettanomyces strain.
 20. The recombinant yeast strain of claim 19, wherein the yeast is Saccharomyces cerevisiae. 21-27. (canceled)
 28. An isolated polynucleotide encoding for a xylose reductase (XR) polypeptide having an amino acid sequence that is at least 85% identical to SEQ ID NO:42 or SEQ ID NO:46.
 29. The polynucleotide of claim 28, wherein the polynucleotide encodes for a xylose reductase (XR) polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO:42 or SEQ ID NO:46.
 30. The polynucleotide of claim 28, wherein the polynucleotide encodes for a xylose reductase (XR) polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO:42 or SEQ ID NO:46.
 31. An isolated polynucleotide encoding for a xylose dehydrogenase (XDH) polypeptide having an amino acid sequence that is at least 85% identical to SEQ ID NO:44 or SEQ ID NO:48.
 32. The polynucleotide of claim 31, wherein the polynucleotide encodes for a xylose reductase (XR) polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO:44 or SEQ ID NO:48.
 33. The polynucleotide of claim 31, wherein the polynucleotide encodes for a xylose reductase (XR) polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO:44 or SEQ ID NO:48. 34-43. (canceled) 